ENZYMOLOGY LABORATORY 1

Instructor: JOHN JEFFREY PANGILINAN, RMT, MSMT LAB 2021 – 2022

nd
Date: February 15, 2022 2 Semester
TRANS 1 CCHM322 LAB
 However, when the substrate concentration reaches a
TOPIC OUTLINE maximal value, higher concentration of substrate no
longer result in increased rate of reaction, saturation
INTRODUCTION kinetics.
FACTORS AFFECTING RATE OF REACTION  Enzyme concentration
SPECIMEN & OTHER CONSIDERATIONS  If substrate is fixed the rate of reaction is determined
MATERIALS NEEDED by the enzyme
AMINOTRANSFERASES (AST & ALT)  The higher the enzyme concentration, the faster is the
 REAGENT reaction, because more enzyme is present to bind with
 PROCEDURES the substrate.
 COMPUTATION  Temperature
AMYLASE - Form of kinetic energy
 CLINICAL APPLICATIONS
 REAGENT & PROCEDURE  An increase in movement and vibrations will increase
 COMPUTATION temperature; Speeding up the movement of particles
LIPASE increases temperature
 CLINICAL APPLICATIONS  An increase in temperature will speed up reaction up to
 MANUAL PROCEDURE a certain point or optimal rate and when it exceeds its
 COMPUTATION specific threshold, denaturation of protein/enzyme due
to excessive heat may occur
 Threshold and optimal rate varies from enzyme to
enzyme
INTRODUCTION O
 37 C is the optimum temperature for enzyme activity
 Increasing temperature usually increases the rate of
Enzymes chemical reaction by increasing the movement of
 They are mostly made up of proteins; this signifies molecules
that Enzymes possess features and properties that  The rate of denaturation increases as the temperature
O O
proteins have. increases, and is usually significant at 40 C to 50 C or
 Enzymes act as a biological catalyst (speeds up the 40 – 45
rate of reaction), but there are a few exemptions,  Optimal pH
where in some enzymes may decelerate rates of  Nature of optimal pH may vary from enzyme to enzyme
reaction  Most physiologic reactions occur in the pH range of 7
 Biological catalysts are important since rates of to 8; certain enzymes may require an acidic, neutral, or
reaction inside the body should be fast enough to alkaline pH
sustain life - Normal Physiologic limit is 7.4 (7.35-7.45)
 Most Enzymes form three dimensional structures with  Extreme pH level may denature an enzyme or
a few exemptions influence its ionic state resulting in structural change or
 In our body Enzymes are not consumed, rather it is change in the charge of amino acid residue in the
continuously recycled active site
- Greatest enzyme substrate complex is 4
FACTORS AFFECTING RATE OF REACTION  pH is determined by amount of hydrogen ions or OH
ions an increase in H ions will make it acidic while an
-The speed at which the rates of reaction occur; “kung gaano increase in OH ions will make it basic
kabagal or kabilis” - incorporate buffer
 Enzyme Substrate Complexes - pH below 7 & greater than 8, IONIZATION is affected
 Forms products and reactions  Inhibitor concentration
 Each biochemical reaction has a specific enzyme  Enzymatic reactions may not progress if an inhibitor
that catalyzes a reaction interferes with the reaction
 Isoenzymes/Isozymes  Competitive, Non-competitive, Uncompetitive inhibition
 Enzymes with small differences in structural  Competitive: Inhibitor will
entity; they have slight modifications per tissue; compete with the
although they have small differences they substrates active site
perform the same reactions  Non-Competitive: Inhibitor
 e.g. Lactate dehydrogenase exists as LDH 1, will attach itself to
LDH 2, LDH 3, LDH 4, LDH 5, LDH 6; they all allosteric site (regulatory
differ in structure/physical form, but perform the site)
same nature of reaction  Uncompetitive: inhibitor
binds itself with the ES
 Substrate concentration complex (enzyme
- Amount or dose substrate)
 Binds to the active site of an enzyme - Need bawasan
 If enzyme is at a fixed concentration, rate of reaction is substrate para di
determined by amount if substrate maggproduce
 With the amount of enzyme exceeding the amount of ESC
substrate, the reaction rate steadily increases as more
substrate is added

H.M. CABRERA & J.R. CAÑETE – TRANSCRIBERS


- Will change only in UNIT OF EXPRESSION
* Reference values varies from places due to different factors
e.g. altitude of laboratory (iba ref. range sa tugeugarao sa
manila) , geographic location, age, gender, reagent
SPECIMEN & OTHER CONSIDERATIONS
 Specimen of choice
 Nonhemolyzed, fresh serum or plasma
** Glycosylated HGB or HBA1C uses WHOLE
BLOOD (EDTA)
 Serum is the specimen of choice since
plasma is more complex and some
anticoagulants may be an interference to
testing
- serum is CLEARER
- Uses RED TOP (30 MINUTES)
- YELLOW TOP (15 MINUTES)
(SST- Serum Separator Tube)
has THIXOTROPIC GEL (clot activator)
 Heparin should be used as anticoagulant (IN
VIVO)if Plasma will be used
- also used as ANTITHROMBIN
 Clotting (FIBRINOGEN) is required before
centrifugation otherwise hemolysis may
occur and may be a source of interference
 Interferences
 Some anticoagulants may be interferences
or inhibit activators such as EDTA since it
chelates calcium it may interfere with
Aminotransferases (AST & ALT)
 RBCs contain Aminotransferases, thus
hemolysis may cause false elevation
 Therapeutic drugs such as MORPHENE and
Opiates may cause false elevation of
amylase due to the constriction of the
spinchter of pancreatic duct which can
increase Amylase in the blood
 TAGS(triglycerides) may inhibit Amylase
- HIGH AMYLASE, HIGH TAGS =still
NORMAL AMYLASE
 Bacteria can falsely elevate Lipase
 Refrigerated samples
- 2-8°C (average of 4-5°C)
- increase shelf life
 Stable for 7 days
 Stable for 3 weeks for lipase; Room temp for
1 week
 Quality control
 Lipase is stable at room temp. for 1 week; 3
weeks if refrigerated, if frozen it is stable for
a few months
 ALT; within 3 days (4°C) it will proceed to
lose 10% of its concentration even if
refrigerated
 The reagents are stable up to the end of the indicated
month of expiry, if stored at 2 – 8°C and
contamination is avoided. Do not freeze the reagents
and store them protected from light!
 If cuvette is not temperature controlled, incubate
samples at 37°C between readings.
 Running controls – High normal, Low normal
 Reagent Quality Control
 Erratic reagent results should be compared
to reagents with the same lot number to
determine deterioration of reagent
 Particulate matter, Cloudiness,
Flocculations, and Precipitations can indicate
reagent deterioration
 Quality control should be compared to high
normal control (upper extreme of reference
value; Example if rev value is 20-40, 40 is
the upper extreme/high control) and low
normal control
2 levels of CONTROL MATERIAL
- LOW NORMAL
- HIGH NORMAL
MATERIALS NEEDED
 Spectrophotometer –based on light performance
 Pipetting device
 Cuvettes
 Interval Timer
 Test tubes
 Test tube rack
 Incubator
 Reagent
 Blood sample
AMINOTRANSFERASES ( AST, ALT )
 AST and ALT are closely related
compounds/enzymes in the Aminotransferases group
 They are responsible for the reversible transfer of
amino acids
**receiver of amino group = KETO ACID
 Reversible, meaning that the reaction is
bidirectional and can go both ways
 AST
 HEART SPECIFIC than ALT
 RBC has 10-15 times amount of AST
 Formerly known as SGOT / GOT (Serum
Glutamic Oxaloacetic Transaminase)
Glutamic & Oxaloacetic = END PRODUCT
 Aspartate aminotransferase (AST) is one of
several enzymes that catalyze the exchange
of amino and oxo groups between alpha-
amino acids and alpha-oxo acids.
 The primary recipient of the Amino group
transferred by AST and ALT are Keto acids
such as Oxaloacetic acid and Pyruvic acid
 Aspartate aminotransferase (AST) is widely
distributed throughout body tissues with
significant amounts in the heart and liver.
Lesser amounts are found in skeletal
muscle, kidneys, pancreas, spleen, lungs,
RBC and brain .
 AST is an early marker of Myocardial
H.M. CABRERA & J.R. CAÑETE – TRANSCRIBERS
 infarction/Heart Attack; AST is a non-specific
marker for both the Heart and Liver, but it is
more specific for the heart
 e.g. Angina pectoris; heart pain. In a heart
attack serum AST will rise within 6-8 hours
and peak within 2 days and decreases within
4-5 days
 This may also happen in Hepatitis,
Liver necrosis, Cirrhosis, and liver
metastasis, but ALT is a more
preferred marker for these liver
diseases

 ALT
 LIVER SPECIFIC than AST
PROCEDURE – AST
 RBC has 5-8 times amount of ALT
 traditionally known as SGPT
GLTUTAMIC & PYRUVATE = END PRODUCT * Make sure that the tubes are clean
1. Prepare AST Working Reagent according to
 Alanine aminotransferase (ALT) is widely
instructions.
reported in a variety of tissue sources. The
2. Zero spectrophotometer at 340 nm with distilled
major source of ALT is of hepatic origin and
water.
has led to the application of ALT
3. For each sample and control, add 1.0 mL Working
determinations to the study of hepatic
Reagent to cuvette or test tube and warm to 37°C for
diseases.
3 minutes.
 ALT is used more for liver diseases such as
*Use a drybath to prewarm the Cuvette
Hepatitis, liver necrosis, cirrhosis, and liver
*Make sure that the Cuvettes are free from scratches which
metastasis
may interfere with the results of the spectrophotometer
 Elevated serum levels are found in hepatitis,
4. Add 100 uL (0.10 mL) serum to its respective tube
cirrhosis, and obstructive jaundice. Levels of
and mix gently.
ALT are only slightly elevated in patients
5. Read and record absorbance at 1 minute. Continue
following a myocardial infarction. (but not
incubating at 37°C and record absorbance again at 2
sensitive)
and 3 minutes.
 Higher concentrations of ALT are found in
*Every succeeding minute for three minutes return the Cuvette
the liver and low concentrations can be
to the drybath after reading the results every minute
found in striated muscles such as cardiac
and skeletal muscles, and in kidneys  AST Principle: Oxidation reaction; Oxaloacetate
 ALT is more liver specific oxidizes NADH to NAD in the presence of MDH
 AST is also referred to as SGOT/ Serum ** OXIDATION
Glutamic Oxaloacetic Transaminase  The speed of Oxidation of NADH to NAD is directly
 ALT is also referred to as SGPT/ Serum proportional to AST activity
Glutamic Pyruvate Transaminase ** NADH –COENZYME ; REDUCED FORM

REAGENT – AST AND ALT

*COMBINATION of R1 and R2 are the working reagents for

both AST and ALT
*Familiarize yourselves with the reagents for ALT and AST
* The Main differences would be Concentrations, L-aspartate
for AST with MDH while ALT uses L-Alanine and doesn’t use
MDH for its working reagent
AST – facilitates the removal of NH2 (Amino group)
from aspartate and combine to 2-Oxoglutarate to
produce L-glutamic acid and Oxaloacetate

PROCEDURE – ALT

*Similar Procedure to AST

1. Prepare ALT Working Reagent according to
**has MALATE DEHYDROGENASE instructions.
2. Zero spectrophotometer at 340 nm with distilled
water.

H.M. CABRERA & J.R. CAÑETE – TRANSCRIBERS

 3. For each sample and control, add 1.0 mL Working
Reagent to cuvette or test tube and warm to 37°C for
3 minutes.
4. Add 100 µL (0.10 mL) serum to its respective tube Computation AST:
and mix gently. *Intial computations should use 4 Decimal Places
5. 5. Read and record absorbance at 1 minute. Continue *The final answer should be 2 Decimal Places
incubating at 37°C and record absorbance again at 2
and 3 minutes. * To know whether the answer is within the
* Pyruvic acid will turn to lactate in the presence of lactate reference value, please refer to the reference
dehydrogenase subsequently reducing NADH to NAD through values table at page 1
Reduction
Computation for Sample:
1 Minute: = 0.0152
2 Minute: = 0.0091
3 Minute: = 0.0065
ALT - catalyze removal of NH2 from alanine (REVERSIBLE =
BIDIRECTIONAL FLOW) and then it will combine to alpha-
0.0152+0.0091+0.0065= 0.0308= =
ketoglutamic acid and will produce PYRUVIC ACID AND
GLUTAMIC ACID
0.0103 x 1768= 18.21U/L
**pyruvic acid is reduced to become LACTATE (with the help
- at 30 the value of the AST sample is within the
of LDH)
reference range
= REDUCTION
- at 37 the value of the AST sample is within the
reference range
COMPUTATION FOR AST/ALT
Computation for High Normal:
 AST Absorvity: 0.00622 (CONSTANT) 1 Minute: = 0.0312
 Constant value of 1768 (MULTIPLIER FACTOR)
2 Minute: = 0.0211
 1768 was derived from 0.10ml (serum) + 1ml (working
reagent = 1.10; 1.10/0.10= 11; 11/0.00622 = 1768 3 Minute: = 0.0163

0.0312+0.0211+0.0163= 0.0686= =

0.0229 x 1768= 40.49U/L

- at 30 the value of the AST sample is above the
reference range
- at 37 the value of the AST sample is above the
reference range
* PS high normal is above the reference range but it is still still
 ALT Absorvity: 0.0063 acceptable since it is not too far away and this was made by
 Constant value of 1746 the manufacturer, otherwise if the sample is above the
 1768 was derived from 0.10ml (serum) + 1ml (working reference range then AST is increased above reference range.
reagent = 1.10; 1.10/0.10= 11; 11/0.0063 = 1746
Computation for Low Normal:
1 Minute: = 0.0022
2 Minute: = 0.0017
3 Minute: = 0.0016

0.0022+0.0017+0.0016= 0.0055= =

ABSORSBANCE Sample CONTROL 0.0018 x 1768= 3.18U/L

(U) - at 30 the value of the AST sample is within the
reference range
High Normal Low Normal
- at 37 the value of the AST sample is within the
reference range
1 – 1 minute 0.0152 0.0312 0.0022
Computation ALT:

2 – 2 minute 0.0181 0.0422 0.0034 Computation for Sample:

1 Minute: = 0.0152
3 – 3 minute 0.0195 0.0489 0.0048 2 Minute: = 0.0091
3 Minute: = 0.0065
0.0152+0.0091+0.0065= 0.0308= =

H.M. CABRERA & J.R. CAÑETE – TRANSCRIBERS

0.0103 x 1746= 17.98U/L
- at 30 the value of the ALT sample is within the
reference range
- at 37 the value of the ALT sample is within the
reference range
*Computation for other parameters (High Normal, Low Normal)
remains the same the difference would only be the constant
(1746)
 AMYLASE
- STARCH AND GLYCOGEN DIGESTION
• Alpha-amylase is an enzyme found in bacteria and
animal tissues that catalyzes the hydrolytic cleavage
of starch and glycogen.
• The acinar cells of the pancreas and the salivary
glands are the major tissue sources of serum AMS.
Lesser concentrations are found in skeletal muscle
and the small intestine and fallopian tubes
CLINICAL APPLICATIONS
• Amylase is also called Diastase
• Amylase belongs to the class of hydrolases (GROUP
3 based on enzyme commission) which means it
requires water in order to split, breakdown, and
catabolize larger complexes into something simpler
• only attacks glycosidic bonds (linkage
for carbohydrates; specifically attacks
the connections of carbon 1 and 4 in order to produce
degeneration products such as maltose, glucose, and
Alpha Limit Dextrin
• 2 types of starch
 Amylose= unbranched form of starch
 Amylopectin= Highly branched starch
• 2 major sources of Amylase
 Salivary Amylase
 Pancreatic Amylase
• Starch Digestion Starts at the mouth (salivary
Amylase) and since salivary amylase has a short
duration it will inactivate (Inhibitory Pathway) with
gastric fluid acidity and the Pancreatic Amylase will
continue the digestion process of starch
• Serum amylase activity determinations are of clinical
interest in the diagnosis of pancreatic function.
• Other disorders causing an elevated serum AMS level
include salivary gland lesions, such as mumps and
parotitis, and other intra-abdominal diseases, such as
perforated peptic ulcer, intestinal obstruction,
cholecystitis, ruptured ectopic pregnancy, mesenteric
infarction, and acute appendicitis. In addition,
elevations have been reported in renal insufficiency
and diabetic ketoacidosis
• Amylase requires calcium and chloride in order to
activate, hence EDTA isn’t a preferred anticoagulant
since it chelates calcium and this will become an
interference
• Amylases can be filtered through normal urine since it
is a very small enzyme
DIAGNOSTIC SIGNIFICANCE
 Serum and Urine Amylase is utilized for the Diagnosis
of Acute Pancreatitis
- required and test for amylase and lipase pag acute
pancreatitis
Pancreatitis (high amylase) = inflammation
Parotitis (high lipase) = inflammation of parotif gland ;
beke
 Amylase is a non-specific test since Amylase can also
be found in other tissues and pancreas
 There are 2 major bands and 4 minor bands of
amylase in electrophoresis
 Normal Serum Bands in Electrophoresis:
o P- band= (Pancreatic type) more common in
urine
 P1
 P2
 P3= is the predominant enzyme in
pancreatitis
o S- band= (Salivary Type = migrates faster in
electrophoresis) 2/3 are present in normal
serum
 S1= Salivary
 S2= Falopian tube
 S3= Lungs
LIPASE – differentiator for amylase to know if salivary or
pancreatic or extra-pancreatic (other than pancreatic such
as salivary, fallopian tube and lungs)
REAGENT AND PROCEDURE
* Has a similar procedure with transaminases; the primary
differences are…
- 405 nm
- 0.025 sample or 25 ul for prewarmed working reagent
- No incubation in between required
- Test results are read every 30 seconds to observe increasing
trend
1. For each sample, add 1.0 mL reagent to cuvette or
test tube and prewarm at 37°C for at least 3 minutes.
2. Zero spectrophotometer with water at 405 nm.
3. Add 0.025 mL (25 µL) serum to its respective tube
and read immediately.
4. Record increase in absorbance at 30 second
intervals for 2 minutes. to know increasing trends of
absorbance
- 0 sec, 30 sec, 60 sec, 90 sec and 120 sec
5. Determine the mean absorbance difference per
minute (∆Abs./min.)
6. Multiply the ∆Abs./min. by 4824 to obtain the result in
U/L.
REAGENT
 HEPES Buffer, pH 7.1 ± 0.1 0.1 mol/L
 Glucosidase (microbial) > 6 KU/L
 Sodium Chloride 62.5 mmol/L
 Magnesium Chloride 12.5 mmol/L
 EPS-G7 (PNPG7) > 8 mmol/L
H.M. CABRERA & J.R. CAÑETE – TRANSCRIBERS
*PNPG7 is hydrolyzed by the amylase converted into PNPG3 +
Maltotetraose
*PNPG3 is hydrolyzed by the glucoamylase converted into ABSORSBANCE Sample
PNPG1 + Glucose (U)
*PNPG1 is liberated by the glucosidase into Glucose + p-
0 – initial 0.0112
Nitrophenol (yellow)
*the intensity of color is directly proportional to the amount of
1 – 30 seconds 0.0134
amylase present in serum
*p-nitrophenol (yellow) is Detectable at 405 nm
2 – 60 seconds 0.0152

3 – 90 seconds 0.0195

4 – 120 seconds 0.0275

How was the constant 4824 derived from the equation

above?

1.025= total volume= 1.0 ml of reagent and 0.025 ml of sample

0.2125= 0.5 (absorvity) x 0.025 (sample volume) x 1.0 (Light
path)

 Amyloclastic
 AKA. Iodometric: Tarch has an iodine
attached component; amylase will act on the
iodin in starch to hydrolase the product
 The Decrease/ Disappearance of color is
proportional to the amylase content of the
particular sample
 Saccharogenic
 Measures reducing sugars; reducing sugars
are directly proportional to the amount of
amylase since reducing sugars e.g. glucose
are degradation products of amylase and
starch
 Chromogenic
 Has attached chromogenic dye
 Color formation
 A higher degree of colorization signifies a
higher degree of amylase, hence colorization
is directly proportional to the amount of
amylase
 Continuous Monitoring
 The oxidation of NADH to NAD is directly
proportional to the amount of amylase a
sample has
 based on oxidation-reduction reaction
AMYLASE COMPUTATION
= 4824
Computation of Amylase Sample:
60 seconds: = 0.0152
120 seconds: = 0.0138
0.0152+0.0138= 0.029= =
0.0145 x 4824= 69.95U/L
- Using serum the value of the Amylase sample is within
the reference range
*Sample Values are checked every 30 seconds to see
increasing trends of values, if values are erratic (meaning;
it goes up and down and it has no pattern) computation is
invalid
 LIPASE
 Pancreas- major source of enzymes
 Lipase is responsible for fat metabolism/ Triglyceride
metabolism
1. Specifically referring to pancreatic Lipase
 Triglyceride/ Triacylglycerol
1. Has a glycerol backbone
2. TAGs that are similar are called Simple
triglycerides
3. TAGs that have variation are called
Complex Triglycerides or Mixed
 Lipases Function as an esterase and is responsible
for the removal and attachment of fatty acids
1. Triglicerides have 3 fatty acids
2. Removal of fatty acids is in a stepwise
manner
I. Diglyceride
II. Monoglyceride
III. Glycerol
3. Fatty acids are the absorbed by the intestinal
wall and reassembled again as triglycerides
4. Triglycerides are then transported and sorted
how the body sees fit
 Lipases are not exclusive to the Pancreas
 Lipase is a group of enzymes, which hydrolyze the

H.M. CABRERA & J.R. CAÑETE – TRANSCRIBERS

 glycerol esters of long chain fatty acids
 The measurement of lipase activity in serum and MANUAL PROCEDURE
other fluid is to evaluate conditions associated with
pancreas  Principle: Since oil is not soluble in water; turbid mixture is
 Voget et al. proposed an olive oil emulsion in formed when added to the buffer when mixed, then
measuring the rate of change in turbidity over a Triglycerides are hydrolyzed when serum is added
specific unit of time. Later Shihabi et al. modified the  Basically the clearer the sample is the lower the
previous method and eliminated some of interference absorbance and the higher the Lipase is, clarity
and Lipase is inversely proportional
CLINICAL APPLICATIONS - HIGH LIPASE = LOW TURBIDITY
• The highest amount of Lipase is found in the  Set the photometer to 0 absorbance with distilled water.
pancreatic tissue, however measurable amounts are  Pipette into a cuvette:
in the GI tract, RBC, and Leukocytes, but it is still a  Mix gently by inversion. Insert cuvette into the cell holder
very useful differentiation for S-amylase/P-amylase as and start stopwatch.
an indicator  Incubate for 1 minute and record initial absorbance
• Clinical assays of serum LPS measurements are reading (0 SEC.).
confined almost exclusively to the diagnosis of acute  Repeat the absorbance readings exactly after 1 and 2
pancreatitis. It is similar in this respect to AMS minutes.
measurements but is considered more specific for  Calculate the difference between absorbances.
pancreatic disorders than AMS measurement. Both  Calculate the mean of the results found for the Blank,
AMS and LPS levels rise quickly, but LPS elevations Sample and Standard to obtain the average change in
persist for approximately 5 days in acute pancreatitis, absorbance per minute (A/min).
whereas AMS elevations persist for only 2 to 3 days.
• An increase in Lipase can signify Chronic Pancreatitis
or obstruction of the pancreatic duct
• Acute Pancreatitis: Lipase increases 2x – 50x folds
of the upper limit within 4-8 hrs and peaks aat 24 hrs;
gradually decreases from 8-14 days
- Upper Limit normal is 150
• e.g. A useful example to differentiate AMS
measurement and LPS measurement is Parotitis and
Pancreatitis
• Parotitis is a disease affecting the salivary
glands while Pancreatitis affects the
pancreas, hence an increase in Amylase and
Lipase may indicate Pancreatitis while an
increase in Amylase only may rule out 400 nm = wavelength for lipase
pancreatitis for Parotitis.
• Lipase Isoenzymes 1.2 ml = working reagent per tube
• L1 **only differ in sample
• L2= is the predominant and the most
clinically significant and specific for COMPUTATION
pancreatitis
• L3

ABSORSBANCE Sample
(U)
Blank Standard
Substrate:
- 0.8% (w/v) olive oil in alcohol
* the most common method is with the use of Olive oil (Gives
of a turbid sample because of fat 0 – initial 0.0112 0.0022 0.0123
 Cherry Crandall method / PRINCIPLE: incubation
of serum with substrate (Olive oil – SOURCE OF
1 – 1 minute 0.0276 0.0034 0.0445
TAGS)
Buffer:
- Tris Buffer 69 mM 2 – 2 minute 0.0389 0.0048 0.0567
- Sodium deoxycholate 10mM
- pH 9.0

H.M. CABRERA & J.R. CAÑETE – TRANSCRIBERS

 = 0.0019
Computation for Sample:
(1 min) 0.0276 – (Intial) 0.0112= 0.0164
Computation for Standard:
(2 min) 0.0389 – (Intial) 0.0112= 0.0277
(1 min) 0.0445 – (Intial) 0.0123= 0.0322
(2 min) 0.0567 – (Intial) 0.0123= 0.0444
0.0164 + 0.0277= 0.0441
= 0.0221 0.0322 + 0.0444= 0.0766
= 0.0383
Computation for Blank:
(1 min) 0.0034 – (Intial) 0.0022= 0.0012
(2 min) 0.0048 – (Intial) 0.0022= 0.0026 = 0.3791 x 85 (Calibrator)= 32.22 U/L

0.0012 + 0.0026= 0.0038 - The value of the Lipase sample is within the reference
= 0.0019 range

*Calibrator value is dependent on the question given in the

Computation for Standard:
quiz; Calibrator can be up to 100
(1 min) 0.0445 – (Intial) 0.0123= 0.0322
(2 min) 0.0567 – (Intial) 0.0123= 0.0444

0.0322 + 0.0444= 0.0766

= 0.0383

= 0.5549 x 50 (Calibrator)= 27.75 U/L

- The value of the Lipase sample is within the reference

range

*If you want to convert U/L to multiply answer to 0.0167

27.75 x 0.0167 = 0.46

*Calibrator value is dependent on the question given in the

quiz

ABSORSBANCE Sample
(U)
Blank Standard

0 – initial 0.0456 0.0022 0.0123

1 – 1 minute 0.0567 0.0034 0.0445

2 – 2 minute 0.0659 0.0048 0.0567

Computation for Sample:

(1 min) 0.0567 – (Intial) 0.0456= 0.0111
(2 min) 0.0659 – (Intial) 0.0456= 0.0203

0.0111 + 0.0203= 0.0314

= 0.0157

Computation for Blank:

(1 min) 0.0034 – (Intial) 0.0022= 0.0012
(2 min) 0.0048 – (Intial) 0.0022= 0.0026

0.0012 + 0.0026= 0.0038

H.M. CABRERA & J.R. CAÑETE – TRANSCRIBERS